Questions related to Innate Immunity
It seems to be an apparent consensus that the activation and function of AhR influences the landscape of TC populations, but is there a group that is more severely affected?
As yet, no one has found the animal that gave people Covid-19. The Center for Disease Control (CDC) points out that at this time, there is no evidence that animals play a significant role in spreading SARS-CoV-2, the virus that causes COVID-19, to people.
SARS-CoV-2 is unprecedented in its combined characteristics: its long period of asymptomatic infection, high transmissibility, significant lethality in high-risk populations, being well-adapted to human cells since its emergence, and having the ability to hijack human innate immunity and bind with high affinity to the human ACE2 receptor.
The reason why we should try hard to figure out the origin of Covid-19 is to inform our efforts to prevent another pandemic like this from happening again. This one was an unfortunate and terrible accident. We should badly want to avoid a second occurrence. We can be blamed for allowing a second one like it if we do not work together soon to find the origin. Right now it appears likely it came directly or indirectly from bats. But specifics would better help us to avoid a second pandemic disaster. Furthermore time is not our friend in finding the origin and sooner is better before information is lost. We need all countries to support a real epidemiological investigation by an unbiased team of scientists given access and authority to take the investigation where ever it leads – possibly to patient zero or to the CoV-2 animal source.
The THP-1 cell showed the classical monocyte (CD14++ CD16-) behavior. If we want to study the non-classical CD14-Cd16++ without drawing blood and isolate the monocyte with FACS, what can we do?
I think, many investigators observed T cell markers on the cells of myeloid lineage (neutrophils, monocytes, dendritic cells, eosinophils, macrophages). Evidently, many of these observations can not be fully explained by artifacts due to co-aggregation with T cells. In my laboratory, we really observed TCR beta and gamma chains on 1-3% of neutrophils and CD4 on CD11c+ (dendritic) cells. In available literature, evidence for expression of rearranged chains of TCR and immunoglobulin molecules in myeloid leukemia cells can be found. My interest to these findings was stressed by recently published works by Kerstin Puellmann and Wolfgang Kaminski with co-authors, showing functionality of recombinatorial immune receptors on neutrophils and macrophages (original publication has appeared in PNAS, 2006, 103(39):14441-6.). However, I see continuation of this history still in one laboratory. My personal doubts are evoked by own practice and the reason that we do not see any support for this idea in responses to allogeneic MHC molecules. Although neutrophils can accumulate in the spleen and blood of animals, immunized with MHC class I disparate tumor cells, they cannot do this without CD8 T cells and do not contain elevated percent of TCR bearing cells. So, I am inclined to think, that T cell markers on myeloid cells represent reminders of developmental history of individual cells, deciding to change lineage too late. I would very appreciate further discussion on this issue.
I know that C57BL/6 mice are naturally resistant to most types of kidney disease models, such as the adriamycin model, however does anyone know if there are models that they are susceptible to? I know they are susceptible to the anti-GBM nephritis model, but I was just wondering if there were any others
Hello! I was reading papers about innate immunity and I encountered this term: "Microorganisms that invade a vertebrate host are initially recognized by the innate immune system through germline-encoded pattern-recognition receptors" ,"A set of germline-encoded receptors, referred to as pattern recognition receptors, evolved in host organisms to recognize PAMPs". But the definition on wiki (In biology and genetics, the germline in a multicellular organism is the population of its bodily cells that are so differentiated or segregated that in the usual processes of reproduction they may pass on their genetic material to the progeny) doesn't seem to fit here.
Thank you in advance.
When I stained mouse PBMCs, I saw there was a population of Ly6c+CD11b- cells (>10%) and these cells express CD86. From the FSC and SSC, I can tell they are not lymphocytes. Does anybody have an idea what kind of cell type these cells are? Enclosed is a figure from a JEM (2012) paper. They could also see this Ly6c+CD11b- population.
MNK 1 & 3 are ILC-like cell lines (Innate Lymphoid Cells) described by Allan DS et al in the article :
An in vitro model of innate lymphoid cell function and differentiation.
Mucosal Immunol. 2015
I was wondering if anyone is currently using these cells because this is the case for me and when I characterized them (stimulation- qPCR, ELISA) (Flow Cytometry), the results are just the opposite of what we're supposed to see.
Any experience or suggestions you'd like to share?
I am trying to find the list of cells that content endosomal TLRs (TLR3,7,8 and9). Are these TLRs expressed all over the body or there are very specific cells that expresses these TLRs?
Any input is appreciated. Any reading material to find the answer is appreciated.
It seems that original wild plants are more resistant to diseases and parasites that those selected and "improved" for their commercial value
I am planning to identify specific innate immunity biomarkers in patients with BKC form tear samples. Can anyone advise which biomarkes are most commonly expressed (e.g. alpha-defensines, beta-defensine-2, cathelicidin (LL-37)) and what methods should I use to measure them (tear sampling)?
I am a fresh man in cell molecular biology. I was studying the liver metabolites function and the inflammatory response with exogenous toxicant. I knew the hepatocyte is the metabolite center for human body. It could synthesize a lot of enzymes, albumin and metabolize toxicant. But I was confused whether the hepatocyte could secrete the pro-inflammatory factor, such as TNF-α, iL-6, when liver or hepatocyte was treated with anti-cancer drug doxorubicin (DOX), and exogenous porphyromonas gingivalis. I saw two papers. They reported the upregulation of mRNA in these treatment.
 Zhang W, Yu J, Dong Q, et al. A mutually beneficial relationship between hepatocytes and cardiomyocytes mitigates doxorubicin-induced toxicity[J]. Toxicology letters, 2014, 227(3): 157-163.
Takano M, Sugano N, Mochizuki S, et al. Hepatocytes produce tumor necrosis factor‐α and interleukin‐6 in response to Porphyromonas gingivalis[J]. Journal of periodontal research, 2012, 47(1): 89-94.
Most of other materials reported that the immune cells and adipose tissue was involved the secretion of these pro-inflammatory factor. It seems the function the hepatocyte only working on the metabolites, enzyme synthesis, and energy storage.
Thank you very much!
Mouse mast cell lines MC/91-14 were initially described by Nabel et al, 1981, Nature, 291(5813), 332-4.
Some authors mention MC/9 cell line others mention MC/9.2 cell line. Are these two different mouse mast cell lines, being the former and latter capable to proliferate after stimulation with IL-4 and IL-10 or IL-10 alone, respectively?
Currently, we know that HSPs can act as a danger associated molecular patter (DAMPs), under stress or immunogenic cell death conditions. When this happens, HSP70 could be released or exposed at the cell membrane, to then be recognized by the PRRs on the innate immune cell surface. But how does a citoplasmatic proteín could be translocated to the cell membrane? Is there any known mechanim that explains this? I think something in the structure of the molecule must change to get in the cell membrane, maybe it becomes more lipophilic. Does anyone knows something about this?
Hi, is there anyone who knows about how IL-6 production is regulated by innate pathways (or other pathways) in the gut under steady state? any difference between small intestine and colon? Thanks!
Is there anyone who has information in regards to ELISA analysis done on crocodilians? Looking to assess parasite load and immune response in crocs, but I can only find one article who's tried this semi-successfully.
Is there a other/better way to measure immune response, or the lack of, in crocodiles in response to parasite load?
I am currently trying to standardise qPCR primers for IL-10 expression in human macrophages i.e. checking amplification, primer efficiencies. Before the actual stimulation by bacteria, I am using control treatments to establish the system using M-CSF mediated THP-1-derived macrophages.
I am using the following treatments:
1. Monocytes only
2. Differentiated but unpolarised Macrophages
3. LPS+ IFNg
5. Glucocorticoids mixture (GCM)
6. As a control for some experiments, PMA-mediated THP1 derived macros.
I have tested the following samples already for the expression of IL-10 and MMP12 with two separate sets of primers.
1. Monos, 2. unpolarised macros 3. LPS + IFNg macros 4. GCM macros
1. I cannot detect IL-10 and MMP12 in monos (Amplified very late Ct 38 or undetermined).
2. Cannot detect IL-10 and MMP12 in M2-like (GCM treated) macros (Amplified very late Ct 38 or undetermined).
3. Very weak MMP12 and almost no IL-10 in M1-like (LPS+IFNg) macros.
1. Should I wait for the TGFbeta1 samples before concluding anything, is it usually expected in this treatment ?
2. Should I check in PMA-treated macros ?
3. What could be a positive control for IL-10 and MMP12 ?
I would be extremely grateful if someone could show me the way here,
I'm having difficulty on getting an 'easy' protocol, that doesn't require many steps and reagents to get the NETs before the SEM processing.
I am trying an antibody that binds specifically with the culturally adapted one. Giving a significant difference between positive and negative controls.
On the other hand, it does not bind specifically with the wild type in splenocytes
Would a good approach be to use supernatants from various breast cancer cell lines and differentiate these CD14+ monocytes from healthy donor blood into macrophages in their presence for 10-14 days? Any other insights?
NLRP3 inflammasome activation results in the release of pro-inflammatory interleukins. Several authors have demonstrated the presence of these interleukins in organs with inflammasome hyperactivation caused by intrinsic or extrinsic damage, for which kidney disease is not the exception; however, inflammasome activation has not been proved to be the cause in the light of an experimental model. Therefore, studying new therapies that focus on removing or inhibiting inflammasome components, both individually and together, is proposed in order to develop the hypothesis raised here. The involvement of inflammasome in human disease has incited efforts to identify potent and specific ways to interfere with NLRP3 activation in the context of auto-inflammatory diseases, including other diseases such as obesity, diabetes and hypertension.
I tried 48 h pre-incubation with rec. m. IFN gamma (should increase IL-31RA at least in human monocyte derived DC). Then stimulation with rec. m. IL-31, but I could not detect any TNF as described for human DC. LPS leads to expected TNF "explosion".
Any ideas? My next step would be IL-6 + low (low!!) dose of LPS.
I plan to check the impact of exogenous cytokines in alteration of M1 or M2 phenotype using mouse macrophages. I'm not familiar with the mouse macrophage cell line. For this purpose, which cell line is suitable? I will determine phenotypes by flow (CD86, IA/IE; M1, MR; M2) and iNOS/Arginase-1 ratio by qPCR.
wondering if anyone knows an easily available human macrophage cell line that we can use to model liver inflammation response mediated by Kuffer cell in vitro? will THP-1 or U937 line be good enough? thanks
Does anyone know what other pathogens we can use in order to derive Cell mediated immune response? or DTH?
It is known that poly I:C stimulates mDC to produce IL12 and IL15, which are potent activators of NK cells. I am wondering if the produced IL12 or IL15 can affect pDC maturation or function. I don't get answers by google. May anyone here share their insights on this ?
When I use a DX5+ Miltenyi kit, the purity is between 50-60%. If I use cell sorting (without any previous separation step), the day after the sorting, NK cells are not functional for cytotoxicity assays (even if they are cultivated with IL-15 1 ng/mL). If I do first DX5+ Miltenyi kit followed by cell sorting, I lose so many cells that I can't do anything.
Leucine-rich repeats are present in a large number of functionally unrelated proteins. All TLR's also contain LRR but recognize different Pathogen-associated molecular patterns, or PAMPs. How does LRR play its role in TLR's and do a number or LRR have any role in function of TLR's?
In mammals, antibodies are classified into five main classes or isotypes – IgA, IgD, IgE, IgG and IgM, according to the heavy chain they contain. Lets say in an effective immunization program, and looking only IgG, IgA and IgM, IgM will first be elicited, which will then undergo isotype switching to IgG and IgA. Why we need different types on antibodies targeting an antigen? Can I say that due to the localisation of the antibodies? For example, IgG is a circulating antibody (abundant in serum) while IgA as mucosal antibody (IgA is the predominant antibody in mucosal secretions)?
Sorry for asking about basics because I am getting confused and get deviated when I read more. I hope there is someone to explain to me a little bit about this. Thanks a lot!
We've been looking a kit to measure the amount of endotoxin in mouse serum (with high-fat diet or not). What's the most reliable and practical kit for this? I found a thread about this but it has been a year. We also looked into the papers and some used QCL-1000 from LONZA. Another new kit is EndoLISA. Does anybody have hands-on experience on any of these? Thanks for your suggestions.
I am aware of RNAi-based therapeutics but am not clear about the natural response of the human body to viruses via RNAi. I remember some article that said RNAi is active at early stages of life and is then slowly replaced by protein-based immunity. I cannot find that paper. I would greatly appreciate a PDF or link to that article or a similar one.
Does anybody has the protocol or information how I can optimize dose of antigen in PBMC culture? How the results should be addressed? Is cell viability result is enough or I should test gene expression level?? My research target is to investigate gene expression. If upto gene expression is necessary, which innate immune genes may be tested??
I'd like to detect pyroptosis and necroptosis in tissue such as skin and skin-draining lymph nodes.
I guess that target cells are most likely epithelial cells or innate immune cells, so isolation and flow cytometric analysis of these cells might not the best way for these cells.
Therefore, I'd like to find ways to probe these types of cell death in tissues. Can you guys recommend me what would be the best markers for these types of cell death, and which analysis (histology, IF and others?) would be the best?
Your kind advise is needed here: I am working with mice- in which we initiate a trauma for inflammation activation. I am planning to block one of the anti-inflammatory cytokines' receptors. My Questions is: Is the activation of the innate immunity after a trauma goes in parallel with the anti-inflammatory cytokines activation? Or in other words, what is the best time to block one of the anti-inflammatory cytokines' receptor after initiating the trauma?
Am thankful already :)
With my Best Reagrds, Alaa
Endotoxin can possibly be neutralized in vivo by diverse substances. The endotoxin- neutalizing substance complex must be removed from the body before it could cause CD14-TLR4-mediated activation of leukocytes and release of inflammatory cytokines leading to ill-effects of endotoxemia. A single mechanism or it will depend on the type of the complex formed?
I am working with macrophage cells and time is of the essence with what I am trying to do. I know that inflammasome formation requires "two hits" of some type of immune stimuli in order to form and for the subsequent secretion of IL-1B, IL-18, and other cytokines. Many people use LPS and ATP for inflammasome formation, but I was curious as to whether or not it was possible to stimulate inflammasome formation in the cells with the "two hits" simultaneously i.e. make a solution of both LPS+ATP. Additionally, what seem to be the best concentrations for both stimuli?
I know MCSFR (CD115 or CSF1R) mRNA is expressed by GMP (granulocyte-macrophage progenitor) and CMP (common-myeloid progenitor), but what about protein? Is CD115 detectable on the cell surface of GMP and CMP?
I was working on C-type lectin-like receptors of the Dectin-1 cluster, especially on CLEC-1, during my PhD project. We tried to find the ligand, but although we tested everything we could get hold of, we couldn't find it.
It seems to me, others might have encountered similar issues. One by one all the other receptors in the family have been characterized thoroughly and there are lots and lots of papers on Dectin-1, CLEC-2, LOX-1, CLEC9a and so on. However, hardly anything on CLEC-1.
I'm working on something very different now, but CLEC-1 is still haunting me...
Is anybody out there still working on it, so that I might find out eventually, what it actually does?
I am interested in the phenotype (very reduced neutrophils and macrophages populations) at 4-5 dpf and was wondering if the morpholinos were efficient that late and if the fish development was normal.
Original paper: Li J, Li K, Dong X, Liang D, Zhao Q (2014) Ncor1 and Ncor2 play essential but distinct roles in zebrafish primitive myelopoiesis. Dev Dyn 243:1544–1553
Innate lymphoid cells (ILCs) are a group of innate immune cells that belong to the lymphoid lineage but do not respond in an antigen-specific manner, as they lack a B or T cell receptor.This relatively newly described group of cells has different physiological functions, some of them analogous to helper T cells, while also including the cytotoxic NK cells. In accordance, they have an important role in protective immunity and the regulation of homeostasis and inflammation, so their dysregulation can lead to immune pathology such as allergy and autoimmune disease.
There's an interaction with a protein, FAP2, on a bacteria, F. Nucleatum, with TIGIT on natural killer cells. I want to target the natural killer cells specifically to block this interaction between the bacteria and the NK cells. I wanted to insert an antibody in mice transgenic for human TIGIT, however if I do so this will elicit an auto-immune response. How can I get around this? I'll consider alternative approaches outside of targeting FAP2 and/or the bacteria.
I am currently working with human NK cell cytotoxicity. We found high expression of perforin and granzyme even in untreated pure NK cell group. It seems like the high expression of perforin and granzymeB have nothing to do with cytotoxicity. I think the trend of CD107a expression must correspond to perforin and granzymeB. But we found CD107a corresponding to the increase of cytotoxicity.
Any suggestion? Any other factors are related to CD107a expression and cytotoxicity?
Integrated blood ROS generation is too time consuming, preferable could be one time point in the begin of blood ROS generation.
I`m trying to activate NLRP3 inflammasome in various cell types with classical method (LPS + ATP).
While I reviewed this process, I noticed that the concentration of ATP I`m using (2mM or 5mM, which is considered as normal to induce NLRP3 inflammasome activation in many papers) is much higher than the concentration that is known to be cytotoxic (less than 100uM...).
I cannot find any difference in viability between control and LPS+ATP treated cells.
How is it possible? Is it LPS priming that protect cells from ATP-induced cytotoxicity? Or Do I misunderstand the inflammasome activation process/ ATP-induced cell damage? Please help me.
I am testing inflammatory properties of a marine sponge extract. this extract gave anti inflammatory activity in first 2 hours with respect to the low doses in carrageenin induced paw oedema test and anti inflammatory activity for leukocyte migration in vivo test (for peritoneal macrophages)using mice, for all the doses. Can this be explainable? Can these two properties present in the same compound (this is an extract which is not fractionated)?
I want to stain mainly the neutrophils and monocytes from the fresh blood. I am looking for any bead based product or any that persevere the blood sample for longer time before stain the innate immune cells.
I am interested in isolating pig monocytes from whole blood. I will do so by Ficoll followed by MACS for CD14 marker.
My 2 questions are: does anyone know the best culture media for maintaining/proliferating adult monocytes, even if not specific for pigs? Also, I want to co-culture these monocytes with plasma I obtained before (kept at -80 since sampling) and evaluate expression of HLA-DR by flow cytometry. Does anyone have an idea of the time I should let the monocytes be co-cultured with the plasma? Should I do step-wise increases in the percentage (vol/vol) of plasma I should add to the culture media prior to deciding how much to add? I dont have a lot of plasma sample from each of the studied animals, so I really need to be cautious with how much I use. I am thinking of using 96 well microplates for culturing so I need little volume from plasmas for the co-cultures.
Any advise will be greatly appreciated.
Thanks a lot.
I am looking for any article highlighting differences between peripheral and central (CNS) inflammatory reaction. With main focus on the mediators.
If mice are given one single dose (0.5mg) of anti-Ly6G (1a8) IP to deplete neutrophils, how long will it take for the neutrophils to be replenished to near normal levels?
I'm evaluating bioactivity of IL12 on different mouse strains splenocytes (C3H/Hej, C57BL/6J, BALB/cByJ). I check IFN gamma production for this evaluation. The results showed that C3H/Hej secretes a ton of IFN gamma, whereas the rest mouse strains didn't do that.
Does anyone know the reason for that phenomena?
Thank you so much for your support!
PolyIC has been used extensively in in vitro experiments to mimic viral infection in cell lines. Can anyone please refer me to an article or publication/document showing that polyIC can be used in vivo (inoculation or injection, etc) to activate antiviral innate immunity.
I know that macrophages reside in most tissues/organs, but I am wondering if anyone is aware of tissues or organs that have relatively high levels of macrophages (in healthy tissue, not during inflammation or other disease)?
For instance, the synovium of the joint has a fairly high percentage of macrophages.
Any other examples?
I've got small issue with isolation of epithelial cells (enterocytes or progenitor cells) from bovine small intestine.
I use chelation method (EDTA+DTT), receive crypts and seed them in rat tail collagen coated flask with DMEM (high glucose) supplemented with EGF, NEAA, T3, hydrocortisone, insulin and 5% FBS.
Next day I can see attached cells around the crypts (island pattern) which is really good. Later on they grow really slow and after 3rd day they detached and died.
What's wrong with my method which is based on another papers?
I'll be really thankful for any suggestions.
I have had experience implanting estrogen pellets (from Innovative Research of America) in nod-scid mice and at week 4 onwards, some form of toxicity and mortality can be observed. My experiments involve MCF-7 xenografts. I am moving on to NSG mice as they are better models for metastasis....hence I am wondering if the same problems (bladder stones etc) still apply to NSG mice.
Also, I have ever read a paper mentioning that estrogen supplementation is not necessary for MCF-7 growth in NSG mice... anyone can confirm this?
Thank you very much in advance!
I have been trying to measure mouse plasma/serum IL-12 by multiplex TH1/TH2 cytokine kit (MSD). And the IL12 concentration from healthy mouse is around 1ng/mL from my measurement. For other cytokines, such as IFN-γ and TNF, they cannot be detected. I tried the IL12p40/p70 ELISA, and it gave me similar result. I am wondering whether this concentration of IL-12 is normal.
Some research studies clearly establish that the elevation of hs-CRP is associated with increased cardiovascular risk, while others fail to find this relationship. Where is the problem of this disagreement?: in the populations studied,in the design of the studies, or in the method of measurement?
Having read this review which I think is perfect, but I am not recognized as immunologist to make such estimations, I am even more convinced that late "Charly" Janeway was a real pioneer. I happily remember his visit at Stanford when he gave a talk in Irv Weissman's house with a huge dinner, a thrilling experience with so fascinating views and scientific as well as social anecdotes. But I guess, we should not only judge good scientist from the awards they received or not.
Is it known which cell type(s) produce 15d-PGJ2 and in response to which stimuli? I'm not very familiar with PG biology and I find the literature on this specific matter a bit confusing and contradictory. Any pointers will be highly appreciated.
Recently I have been working with detecting serum IL12 by ELISA. Based on that result, the sera from healthy mice have around 10000~20000pg/mL IL12p40/p70. But when I searched the literature, IL-12p40 is around 200pg/mL. The coating antibody and detection antibody I used can both recognize p40 and p70. The standard curve looked fine. I diluted the serum by 30 fold, and I could still detect the OD signal. And the negative control (buffer alone) had a low background.
I am wondering whether it is because the antibody can detect both p40 and p70. Still, the concentration of p40 plus p70 is too high. And does anybody know what specific antibody should I use to only detect IL12?
Going back as far as I can remember, people have been to-and-fro about the benefit or deficit of an immune or inflammatory response in human malignant disease and/or metastatic progression. Tumour-Associated Lymphocytes (eg Rosenberg), Tumour Associated Macrophages (eg Fidler), Melanoma-Specific Antigens, CD4/CD8 ratios, Th17/Tregs, IL-10, gamma-deltas, MDSCs, Antigen-Pulsed IFN-primed DCs (eg Kalinski), uncontrolled systemic inflammation (eg McMillan)... the list goes on.
SO my question is - (and yes, I can use Pubmed and read Nature Reviews as well as anyone) - for those of you who are research active in this field - experimentally and clinically - is a consensus emerging for the common solid tumours in particular?
* Does immune/inflammation support early tumour growth?
* Are patients immunologically tolerant to their tumours?
* Do inflammatory phenomena promote metastatic spread and/or seeding?
* How do the murine models - including immune reconstitution models - reflect human disease?
* What are the mistakes and deadends that get repeated with each new generation of researchers?
If there is sufficient interest and/or controversy in this area, then I'm going to commission a special issue of "Genes and Immunity" on the topic.
I wanted to test my cells ROS production by Reactive Oxygen Species (ROS) Detection Reagents (invitrogen), but I did not get positive results even with my control (PMA treated), ROS is lower than unstained cells. The reagent is not the problem because someone used before and got results.
The PMA treated I used is 50uM for 4 hours and while using flow cytometer, the living cells is enough for detection.